Cohesive osteogenic putty and materials therefor

ABSTRACT

Described is an implantable medical material comprising a malleable, cohesive, shape-retaining putty including mineral particles, insoluble collagen fibers and soluble collagen. The medical material can be used in conjunction with biologically active factors such as osteogenic proteins to treat bone or other tissue defects in patients.

BACKGROUND OF THE INVENTION

The present invention relates generally to medical putty implantmaterials, and in certain aspects to collagenous medical putty implantmaterials.

A variety of materials have been suggested for the treatment of bonedefects. In addition to traditional bone grafting, a number of syntheticbone graft substitutes have been used or explored, including severalputty materials.

To conduct bone through-growth effectively, implant materials derivebenefit from the presence of substantial scaffolding material such asbiocompatible ceramics or other mineral scaffolds. Such mineralmaterials are generally hard, brittle substances. The incorporation ofsubstantial levels of mineral particles into putty materials,particularly in respect of granules or other relatively large particles,proves difficult because the large pieces of hard mineral tend todisrupt the putty mass such that it is readily broken or eroded away,and lacks cohesiveness desired for handling prior to implant and forpersistence after implant. This may present problems in achievingeffective bone growth into and through the desired implant volume, dueto migration or separation of the scaffolding particulates.

In view of the background in the area, there exist needs for improvedputty materials which not only have high levels of incorporated,relatively large mineral particles, but also maintain the desiredcombination of malleability and cohesiveness. In certain aspects, thepresent invention is directed to these needs.

SUMMARY OF THE INVENTION

Accordingly, in one aspect, the present invention is directed to animplantable osteogenic medical material comprising a malleable,cohesive, shape-retaining putty that includes a combination of mineralparticles and collagen, wherein the collagen includes insoluble collagenfibers and an equal or relatively lesser amount by weight of solublecollagen. Thus, in one embodiment, the invention provides a malleable,cohesive, shape-retaining putty that comprises 60% to 75% by weight ofan aqueous liquid medium and includes a bone morphogenic proteinincorporated at a level of about 0.6 milligrams per cubic centimeter(mg/cc) to about 2 mg/cc. The putty includes mineral particles dispersedtherein having an average particle diameter in the range of 0.4millimeters (mm) to 5 mm at a level of 0.25 g/cc to 0.35 g/cc in theoverall putty. The putty further includes insoluble collagen fibers at alevel of 0.04 g/cc to 0.1 g/cc, and soluble collagen at a level of 0.01g/cc to 0.08 g/cc, with the further proviso that the weight ratio ofinsoluble collagen fibers to soluble collagen in the putty is in therange of 4:1 to 1:1.

In another embodiment, the invention provides an implantable medicalmaterial comprising a malleable, cohesive, shape-retaining puttycomprised 60% to 75% by weight of an aqueous liquid medium, anddispersed mineral particles having an average particle diameter in therange of 0.4 mm to 5 mm at a level of 0.25 g/cc to 0.35 g/cc. The puttyalso includes insoluble collagen fibers at a level of 0.04 g/cc to 0.1g/cc, and soluble collagen at a level of 0.01 g/cc to 0.08 g/cc, withthe proviso that the weight ratio of insoluble collagen fibers tosoluble collagen is in the range of 4:1 to 1:1. Such putty can be usedas an osteoconductive material, for example in bone void fillerapplications, and/or can be modified to incorporate one or moreosteogenic proteins to provide an osteogenic putty.

In another embodiment, the invention provides a method for preparing animplantable medical putty material. The method includes providing a dry,porous material that includes a particulate mineral material having anaverage particle diameter of about 0.4 mm to about 5 mm embedded withina disruptable collagenous matrix. The dried material is comprised 70% to90% by weight of the particulate ceramic material and 10% to 30% byweight of collagen. The collagenous matrix includes insoluble collagenfibers and soluble collagen present in a weight ratio of 4:1 to 1:1. Themethod includes the further step of applying an amount of aqueous mediumto the dried material and disrupting the collagenous matrix so as toprepare a malleable, cohesive, shape-retaining putty that comprises 60%to 75% by weight of water. In certain aspects of this embodiment, theaqueous medium can include a bone morphogenic protein dissolved thereinat a level of about 0.6 mg/cc to about 2 mg/cc, so as to result in animplantable osteogenic medical material.

In still another embodiment, the invention provides an implant materialthat comprises a dried porous body including a particulate ceramicmaterial having an average particle diameter of 0.4 mm to 5 mm embeddedwithin a disruptable collagenous matrix, wherein the body comprises 70%to 90% by weight of the particulate mineral material and 10% to 30% byweight of collagen. The collagenous matrix includes insoluble collagenfibers and soluble collagen, wherein the insoluble collagen fibers andsoluble collagen are present in a weight ratio of 4:1 to 1:1. The driedporous body is wettable with a biocompatible aqueous liquid to form amalleable, cohesive, shape-retaining putty material that includes anadmixture of the collagen fibers, aqueous collagen gel, and theparticulate mineral material.

In still further embodiments, the present invention provides methods fortreating patients that involve implanting in the patients a medicalmaterial as described herein.

Additional embodiments as well as features and advantages of the presentinvention will be apparent to those of ordinary skill in the art fromthe descriptions herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a perspective view of a dried porous body implantmaterial of the invention including a reservoir for receipt of a wettingliquid.

FIG. 2 provides a perspective view of a cylindrical-form dried porousbody implant material of the invention.

FIG. 3 provides a chart showing the average flexibility of the normalintact and two treated groups in flexion and extension loads, asdescribed in Example 2 below.

FIG. 4 provides a chart showing the average flexibility of the normalintact and two treated groups in right and left axial rotation loads, asdescribed in Example 2 below.

FIG. 5 provides a chart showing the average flexibility of the normalintact and two treated groups in right and left lateral bending loads,as described in Example 2 below.

FIG. 6 provides a chart showing the average stiffness of the intact andtreated animal groups under flexion and extension loads, as described inExample 3 below.

FIG. 7 provides a chart showing the average stiffness of the intact andtreated animal groups under axial rotation loads, as described inExample 3 below.

FIG. 8 provides a chart showing the average stiffness of the intact andtreated animal groups under lateral bending loads, as described inExample 3 below.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of theinvention, reference will now be made to certain embodiments andspecific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of theinvention is thereby intended, such alterations and furthermodifications in the illustrated device, and such further applicationsof the principles of the invention as described herein beingcontemplated as would normally occur to one skilled in the art to whichthe invention relates.

As disclosed above, in certain aspects, the present invention relates toimplantable osteogenic medical putty, and to methods and materials thatare useful for preparing such an osteogenic medical putty. Preferredmedical putty materials of the invention possess a combination ofadvantageous properties including high mineral content, malleability,cohesiveness, and shape retention. In this regard, as used herein theterm “malleable” means that the material is capable of being permanentlyconverted from a first shape to a second shape by the application ofpressure. The term “cohesive” as used herein means that the putty tendsto remain a singular, connected mass upon stretching, including theexhibition of the ability to elongate substantially without breakingupon stretching. In the context of putties of the invention containinginsoluble collagen fibers and soluble collagen, upon stretching, theadvantageous putties exhibit elongation, during which the existence ofsubstantial levels of intermeshed collagen fibers clinging to oneanother becomes apparent. As used herein, the term “shape-retaining”means that the putty material is highly viscous and unless acted uponwith pressure tends to remain in the shape in which it is placed. Thisis contrasted to thinner paste form materials which readily flow, andthus would pool or puddle upon application to a surface. In certainfeatures of the invention, novel combination of ingredients provide amedical putty material that not only contains a significant, high levelof large particulate mineral particles, but also exhibits superiorproperties with respect to malleability, cohesiveness, and shaperetention.

Putties according to aspects of the present invention will include acombination of soluble collagen and insoluble collagen. “Solublecollagen” refers to the solubility of individual tropocollagen moleculesin acidic aqueous environments. Tropocollagen may be considered themonomeric unit of collagen fibers and its triple helix structure is wellrecognized. “Insoluble collagen” as used herein refers to collagen thatcannot be dissolved in an aqueous alkaline or in any inorganic saltsolution without chemical modification, and includes for example hides,splits and other mammalian or reptilian coverings. For example, “naturalinsoluble collagen” can be derived from the corium, which is theintermediate layer of an animal hide (e.g. bovine, porcine, etc.) thatis situated between the grain and the flesh sides. “Reconstitutedcollagen” is essentially collagen fiber segments that have beendepolymerized into individual triple helical molecules, then exposed tosolution and then reassembled into fibril-like forms.

The collagen putty of the preferred embodiment of the present inventioncontains both soluble collagen and insoluble collagen fibers. Thesoluble collagen and insoluble collagen fibers can first be preparedseparately, and then combined. Both the soluble collagen and theinsoluble collagen fibers can be derived from bovine hides, but can alsobe prepared from other collagen sources (e.g. bovine tendon, porcinetissues, recombinant DNA techniques, fermentation, etc.).

In certain embodiments, the invention provides putty-form compositionsthat include the insoluble collagen fibers at a level of 0.04 g/cc to0.1 g/cc of the putty, and soluble collagen at a level of 0.01 g/cc to0.08 g/cc of the putty. In other embodiments, such compositions includeinsoluble collagen fibers at a level of about 0.05 to 0.08 g/cc in theputty, and soluble collagen at a level of about 0.02 to about 0.05 g/ccin the putty. In general, putties of the invention will includeinsoluble collagen fibers in an amount (percent by weight) that is atleast equal to or greater than the amount of soluble collagen, tocontribute beneficially to the desired handling and implant propertiesof the putty material. In advantageous embodiments, the collagenousmatrix will include insoluble collagen fibers and soluble collagenpresent in a weight ratio of 4:1 to 1:1, more advantageously about 75:25to about 60:40. Further still, additional desired putties of theinvention include the insoluble collagen fibers and soluble collagen ina weight ratio of about 75:25 to about 65:35, and in one specificembodiment about 70:30.

Medical putties of the present invention also include an amount of aparticulate mineral material. In certain aspects of the invention, theparticulate mineral is incorporated in the inventive putty compositionat a level of at least about 0.25 g/cc of putty, typically in the rangeof about 0.25 g/cc to about 0.35 g/cc. Such relatively high levels ofmineral will be helpful in providing a scaffold for the ingrowth of newbone.

The mineral used in the present invention can include a natural orsynthetic mineral that is effective to provide a scaffold for boneingrowth. Illustratively, the mineral matrix may be selected from one ormore materials from the group consisting of bone particles, Bioglass®,tricalcium phosphate, biphasic calcium phosphate, hydroxyapatite,corraline hydroxyapatite, and biocompatible ceramics. Biphasic calciumphosphate is a particularly desirable synthetic ceramic for use in theinvention. Such biphasic calcium phosphate can have a tricalciumphosphate:hydroxyapatite weight ratio of about 50:50 to about 95:5, morepreferably about 70:30 to about 95:5, even more preferably about 80:20to about 90:10, and most preferably about 85:15. The mineral materialcan be particulate having an average particle diameter between about 0.4and 5.0 mm, more typically between about 0.4 and 3.0 mm, and desirablybetween about 0.4 and 2.0 mm.

In another aspect of the invention, the mineral material can includebone particles, possibly cancellous but preferably cortical, ground toprovide an average particle diameter among those discussed above for theparticulate mineral material. Both human and non-human sources of boneare suitable for use in the instant invention, and the bone may beautographic, allographic or xenographic in nature relative to the mammalto receive the implant. Appropriate pre-treatments known in the art maybe used to minimize the risks of disease transmission and/or immunogenicreaction when using bone particles as or in the mineral material.

In one embodiment, xenogenic bone that has been pretreated to reduce orremove its immunogenicity is used in or as the porous mineral matrix inthe implant composition. For example, the bone can be calcined ordeproteinized to reduce the risks of immunogenic reactions to theimplant material.

A putty-form composition of the invention can include a significantproportion of a liquid carrier, which will generally be an aqueousliquid such as water, saline, or buffered solutions. In one aspect, amalleable, cohesive, shape-retaining putty of the invention comprisesabout 60% to 75% by weight of an aqueous liquid medium, such as water,advantageously about 65% to 75% by weight of an aqueous liquid medium(e.g. water).

Putty-form compositions can also include a bone morphogenic proteinincorporated therein in an effective amount to render the puttyosteogenic when implanted in a mammal, such as a human patient. In oneembodiment, an inventive putty composition includes bone morphogenicprotein at a level of about 0.6 milligrams per cubic centimeter (mg/cc)of putty to about 2 mg/cc of putty, advantageously at a level of about0.8 mg/cc to about 1.8 mg/cc.

As noted above, the compositions of the invention will generallyincorporate at least as much insoluble collagen fiber as solublecollagen on a weight basis, e.g. in a weight ratio of about 4:1 to about1:1. Advantageous putty compositions will include more insolublecollagen fibers than soluble collagen, for instance, in a weight ratioof about 75:25 to about 60:40, more desirably about 75:25 to about65:35, and in one specific embodiment about 70:30. Suitable collagenmaterials for these purposes can be prepared using techniques known inthe literature or can be obtained from commercial sources, including forexample from Kensey Nash Corporation (Exton, Pa.) which manufacturessoluble collagen known as Semed S, fibrous collagen known as Semed F,and a composite collagen known as P1076.

Any suitable osteogenic material can be used in methods and/orcompositions of the invention, including for instance harvestedautologous bone or other suitable osteogenic substances. In certainembodiments, the osteogenic substance can include a growth factor thatis effective in inducing formation of bone. Desirably, the growth factorwill be from a class of proteins known generally as bone morphogenicproteins (BMPs), and can in certain embodiments be recombinant human(rh) BMPs. These BMP proteins, which are known to have osteogenic,chondrogenic and other growth and differentiation activities, includerhBMP-2, rhBMP-3, rhBMP4 (also referred to as rhBMP-2B), rhBMP-5,rhBMP-6, rhBMP-7 (rhOP-1), rhBMP-8, rhBMP-9, rhBMP-12, rhBMP-13,rhBMP-15, rhBMP-16, rhBMP-17, rhBMP-18, rhGDF-1, rhGDF-3, rhGDF-5,rhGDF-6, rhGDF-7, rhGDF-8, rhGDF-9, rhGDF-10, rhGDF-11, rhGDF-12,rhGDF-14. For example, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7,disclosed in U.S. Pat. Nos. 5,108,922; 5,013,649; 5,116,738; 5,106,748;5,187,076; and 5,141,905; BMP-8, disclosed in PCT publicationWO91/18098; and BMP-9, disclosed in PCT publication WO93/00432, BMP-10,disclosed in U.S. Pat. No. 5,637,480; BMP-11, disclosed in U.S. Pat. No.5,639,638, or BMP-12 or BMP-13, disclosed in U.S. Pat. No. 5,658,882,BMP-15, disclosed U.S. Pat. No. 5,635,372 and BMP-16, disclosed in U.S.Pat. Nos. 5,965,403 and 6,331,612. Other compositions which may also beuseful include Vgr-2, and any of the growth and differentiation factors[GDFs], including those described in PCT applications WO94/15965;WO94/15949; WO95/01801; WO95/01802; WO94/21681; WO94/15966; WO95/10539;WO96/01845; WO96/02559 and others. Also useful in the present inventionmay be BIP, disclosed in WO94/01557; HP00269, disclosed in JPPublication number: 7-250688; and MP52, disclosed in PCT applicationWO93/16099. The disclosures of all of these patents and applications arehereby incorporated herein by reference. Also useful in the presentinvention are heterodimers of the above and modified proteins or partialdeletion products thereof. These proteins can be used individually or inmixtures of two or more. rhBMP-2 is preferred.

The BMP may be recombinantly produced, or purified from a proteincomposition. The BMP may be homodimeric, or may be heterodimeric withother BMPs (e.g., a heterodimer composed of one monomer each of BMP-2and BMP-6) or with other members of the TGF-beta superfamily, such asactivins, inhibins and TGF-beta 1 (e.g., a heterodimer composed of onemonomer each of a BMP and a related member of the TGF-beta superfamily).Examples of such heterodimeric proteins are described for example inPublished PCT Patent Application WO 93/09229, the specification of whichis hereby incorporated herein by reference. The amount of osteogenicprotein useful herein is that amount effective to stimulate increasedosteogenic activity of infiltrating progenitor cells, and will dependupon several factors including the size and nature of the defect beingtreated, and the carrier and particular protein being employed. Incertain embodiments, the amount of osteogenic protein to be deliveredwill be in a range of from about 0.05 to about 1.5 mg.

Other therapeutic growth factors or substances may also be used inputties of the present invention, especially those that may be used tostimulate bone formation. Such proteins are known and include, forexample, platelet-derived growth factors, insulin-like growth factors,cartilage-derived morphogenic proteins, growth differentiation factorssuch as growth differentiation factor 5 (GDF-5), and transforming growthfactors, including TGF-α and TGF-β. As well, other biologically-derivedmatrix materials such as demineralized bone matrix (DBM) may beincorporated into putties of the invention.

The osteogenic proteins or other biologically active agents to be usedin the present invention can be provided in liquid formulations, forexample buffered aqueous formulations. In certain embodiments, suchformulations are mixed with, received upon and/or within, or otherwisecombined with a dried implant material in order to prepare an osteogenicputty material of the invention.

As further enhancements of the compositions of the present invention,those skilled in the art will readily appreciate that other osteogenicenhancing factors may be incorporated into the composition. Suchadditional factors include, but are not limited to host compatibleosteogenic progenitor cells, autographic bone marrow, allographic bonemarrow, transforming growth factor-beta, fibroblast growth factor,platlet derived growth factor, insulin-like growth factor,microglobulin-beta, antibiotics, antifungal agents, wetting agents,glycerol, steroids and non-steroidal anti-inflammatory compounds.

In another aspect, the present invention provides a dried implantmaterial that can be combined with an appropriate amount of an aqueousmedium in order to prepare putty materials of the invention. The driedimplant material will be a porous body that includes a particulatemineral material having an average particle diameter of about 0.4 mm toabout 5.0 mm embedded within a disruptable collagenous matrix. Thedried, porous implant material will be comprised 70% to 90% by weight ofthe particulate mineral material and 10% to 30% by weight of collagen.The collagenous matrix will include insoluble collagen fibers andsoluble collagen present in a weight ratio as discussed above, that is,4:1 to 1:1, advantageously about 75:25 to about 60:40, moreadvantageously about 75:25 to about 65:35, and in one specificembodiment about 70:30. In addition, as discussed above, the particulatemineral material will typically have an average particle diameterbetween 0.4 and 3.0 mm, and more desirably between 0.4 and 2.0 mm.

The dried, porous body can have a density of between about 0.1 g/cc toabout 0.3 g/cc, more desirably between about 0.15 g/cc and about 0.25g/cc, and in certain aspects between about 0.18 g/cc and about 0.25g/cc. Such dried, porous implant bodies can also exhibit porosities ofat least about 50%, more desirably at least about 70% up to about 90%,and in certain embodiments in the range of about 80% to about 90%.

The dried, porous implant bodies in accordance with the invention can beprovided in any suitable shape, including cylinders, cubes, or othershapes. In certain aspects, the dried, porous implant body can define areservoir for receiving amounts of a wetting liquid, e.g. to be used inthe preparation of a putty from the dried implant material.

The dried porous body can be prepared using any suitable technique,including for example casting a liquid medium containing the dryingredients, and then drying that medium by any appropriate means suchas air drying or lyophilization.

With reference to FIG. 1, depicted is an illustrative, dried porousimplant body 11 of the invention. The body 11 has a cast material 12which defines a reservoir 13. The reservoir 13 has a bottom surface 14and side walls 15, and can hold a liquid to be used to wet the body 11in the formation of a putty, such that the liquid can be convenientlycharged to reservoir 13 and allowed to soak into body 11 over time. Thebody 11 has an upper surface 16 surrounding the reservoir 13, as well assidewalls 17 and a bottom surface 18.

Referring to FIG. 2, shown is another dried, porous implant body 21according to one embodiment of the invention. Implant body 21 iscylindrical in shape, having a circular cross section. Implant body 21is thus conveniently shaped for receipt within a cylindrical syringebarrel of a roughly corresponding or greater cross sectional dimension.Implant body 21 includes a first end 22, a second end 23, and a smooth,rounded external surface 24.

In use, the dried, porous body (e.g. 11, 21) can be combined with asufficient amount of a liquid material such as an aqueous medium toprepare a putty form material as described herein. The body (11, 21)will exhibit a disruptable character, such that it can be broken down byphysical manipulation (e.g. manual crushing and kneading) to form aputty when combined with a liquid. Thus, chemical, covalent crosslinking between the collagen materials in the dried material, if any,will generally be minimal. Other modes of providing integrity to thebody (11, 21) can be used, e.g. dehydrothermal cross linking, or crosslinking or adhesive forces imparted by ionic or hydrogen bonding. Itwill thus be understood that cross linking can be present in the dried,porous body (11, 21), but that it will be of such a nature to leave thebody disruptable to form a putty as described herein.

Typically, the combination of a dried porous implant body of theinvention with the liquid carrier, and the physical kneading or othermixing of the resultant mass, will result in a reduction of the volumeof the dried porous body, for example resulting in a putty volume thatis about 30% to about 70% of that of the original implant body, moretypically about 40% to about 60%. This is a result of a breakdown of theoriginal porosity of the dried implant body to form a relatively lessporous or non-porous putty implant composition. The liquid carrier willtypically be an aqueous substance, including for instance sterile water,physiological saline, blood, bone marrow, bone marrow fractions or othersolutions (with or without organic co-solvents), emulsions orsuspensions that provide adequate wetting characteristics to formputties of the invention.

In use, the putty implant compositions of the invention are implanted ata site at which bone growth is desired, e.g. to treat a disease, defector location of trauma, and/or to promote artificial arthrodesis. Theputty form of the compositions enables their positioning, shaping and/ormolding within voids, defects or other areas in which new bone growth isdesired. In particularly advantageous embodiments, the shape-retainingproperty of the putty material of the invention will desirably providesufficient three-dimensional integrity to resist substantial compressionwhen impinged by adjacent soft tissues of the body at a bony implantsite.

Bone repair sites that can be treated with medical putty compositions ofthe invention include, for instance, those resulting from injury,defects brought about during the course of surgery, infection,malignancy or developmental malformation. The putty compositions can beused in a wide variety of orthopedic, periodontal, neurosurgical andoral and maxillofacial surgical procedures including, but not limitedto: the repair of simple and compound fractures and non-unions; externaland internal fixations; joint reconstructions such as arthrodesis;general arthroplasty; cup arthroplasty of the hip; femoral and humeralhead replacement; femoral head surface replacement and total jointreplacement; repairs of the vertebral column including spinal fusion andinternal fixation; tumor surgery, e.g., deficit filing; discectomy;laminectomy; excision of spinal cord tumors; anterior cervical andthoracic operations; repairs of spinal injuries; scoliosis, lordosis andkyphosis treatments; intermaxillary fixation of fractures; mentoplasty;temporomandibular joint replacement; alveolar ridge augmentation andreconstruction; inlay osteoimplants; implant placement and revision;sinus lifts; cosmetic enhancement; etc. Specific bones which can berepaired or replaced with the isolate or implant comprising the isolateinclude, but are not limited to: the ethmoid; frontal; nasal; occipital;parietal; temporal; mandible; maxilla; zygomatic; cervical vertebra;thoracic vertebra; lumbar vertebra; sacrum; rib; sternum; clavicle;scapula; humerus; radius; ulna; carpal bones; metacarpal bones;phalanges; ilium; ischium; pubis; femur; tibia; fibula; patella;calcaneus; tarsal and metatarsal bones.

Once in place, osteogenic putty form implant compositions of theinvention can effectively induce and support the ingrowth of bone intothe desired area even in a primate subject such as a human exhibiting arelatively slow rate of bone formation compared to smaller mammals, forexample rodents or rabbits.

Osteogenic putty compositions of the invention are especiallyadvantageous when used in bones or bone portions that are vascularizedto only moderate or low levels. These areas present particularly lowrates of bone formation, and as such the rapid resorption of the carrierposes enhanced difficulties. Examples of moderate or only slightlyvascularized sites include, for example, transverse processes or otherposterior elements of the spine, the diaphysis of long bones, inparticular the mid diaphysis of the tibia, and cranial defects. Anespecially preferred use of paste compositions of the invention is as animplant to promote arthrodesis between vertebrae in spinal fusions inhumans or other mammals, including for example interbody, posteriorand/or posterolateral fusion techniques. Illustratively, as set forth inExample 3 below, osteogenic putty compositions containing recombinanthuman BMP-2 were used successfully in posterolateral fusion procedures,achieving highly beneficial arthodesis as examined by both radiographicand biomechanical techniques, comparing very favorably to autograft bone(long considered the “gold standard” in such fusions).

In addition, in accordance with other aspects of the invention, theputty compositions of the invention can be incorporated in, on or arounda load-bearing spinal implant (e.g. having a compressive strength of atleast about 10000 N) implant device such as a fusion cage, dowel, orother device having a pocket, chamber or other cavity for containing anosteogenic composition, and used in a spinal fusion such as an interbodyfusion. One illustrative such use is set forth in Example 2 below, inwhich an inventive osteogenic putty was used in conjunction with aload-bearing interbody spinal spacer to achieve a highly successfulinterbody fusion, again comparing very favorably to autograft bone.

Medical putty compositions of the present invention can also be used incombination with cells, including for instance progenitor and/or stemcells derived from embryonic or adult tissue sources and/or taken fromculture. Illustratively, putties of the invention can incorporate cellsderived from blood, bone marrow, or other tissue sources from thepatient to be treated (autologous cells) or from a suitable allogenic orxenogenic donor source. In certain embodiments of the invention, puttiesof the invention incorporate an enriched bone marrow fraction, preparedfor example as described in US Patent Publication No. 2005/0130301 toMcKay et al. published Jun. 16, 2005, publishing U.S. patent applicationSer. No. 10/887,275 filed Jul. 8, 2004, which is hereby incorporatedherein by reference in its entirety. Thus, putty materials canincorporate a bone marrow fraction enriched in connective tissue growthcomponents, that is prepared by centrifuging a biological sample (e.g.from the patient to be treated) to separate the sample into fractionsincluding a fraction rich in connective tissue growth components. Thefraction rich in connective tissue growth components can then beisolated from the separated sample, and incorporated into the puttymaterial of the present invention, e.g. by using the fraction in or asthe wetting medium for the dried, porous body as discussed hereinabove.

The present invention also provides medical kits that can be used toprepare implant compositions. Such kits can include a dried, porous bodyaccording to the invention, along with an aqueous medium for combinationwith the body to form a putty and/or another item such as a load-bearingimplant (e.g. a spinal spacer) and/or an osteogenic substance such as aBMP. In one specific form, such a medical kit will include the dried,porous body, a BMP in lyophilized form (e.g. rhBMP-2), and an aqueousmedium for reconstitution of the BMP to prepare an aqueous formulationthat can then be combined with the dried, porous body to prepare anosteogenic putty of the invention.

The invention will now be more particularly described with reference tothe following specific Examples. It will be understood that theseExamples are illustrative and not limiting of the invention.

Example 1 Preparation of Inventive Putty with rhBMP-2

9 ml of a buffered aqueous solution of rhBMP-2 (1.5 mg/ml solution, asavailable with INFUSE® Bone Graft, Medtronic Sofamor Danek, Memphis,Tenn.) were added to a dried, porous cylindrical body having a volume of18 cc and weighing approximately 3.8 grams. The dried, porous body hadbeen prepared by casting and then lyophilizing an aqueous suspension ofinsoluble collagen fibers, acid soluble collagen, and ceramic granules,exhibited a porosity of about 85%, and was comprised of the following:

Material Wt % Solids Biphasic CaP Granules* 80% Insoluble CollagenFibers 14% Acid Soluble Collagen  6% *Mastergraft ® Ceramic Granules,biphasic calcium phosphate granules containing 85% tricalcium phosphateand 15% hydroxyapatite, particle size 0.5-1.6 mm,After soaking, the implant material and added rhBMP-2 solution werethoroughly mixed by kneading to prepare approximately 10 cc of animplantable putty material comprised about 70% by weight of water andcontaining about 0.3 g/cc biphasic calcium phosphate ceramic granules,0.05 g/cc insoluble collagen fibers, 0.02 g/cc acid soluble collagen,and 1.5 mg/cc of rhBMP-2. The resulting osteogenic putty exhibitedsuperior properties for handling and use. The putty retained its shapeunless acted upon, and formed a malleable, cohesive, fibrous mass withentrained granules, that would elongate without breaking uponstretching.

Example 2 Use of Inventive Putty in Interbody Spinal Fusions

An ovine interbody fusion model was used to compare the ability of a 5mm×11 mm×11 mm polyetheretherketone spinal spacer (VERTE-STACK®CORNERSTONE® PSR PEEK Implant, Medtronic Sofamor Danek, Memphis, Tenn.)with packed-in Autograft and the PEEK spacer with packed-in InventivePutty of Example 1 (IP+rhBMP-2) to effect interbody fusion at 6 monthspost-operatively. The efficacy of these treatments to induce interbodyfusion in the ovine lumbar fusion model using blinded radiographic,biomechanical, and histologic measures was evaluated. Assessment offusion was made with Faxitron high-resolution radiography,non-destructive biomechanical testing, and undecalcified histology withcorresponding microradiography. All analyses were conducted in a blindedfashion. In addition, undecalcified histology was used to evaluate theosteocompatibility of the Inventive Putty. In addition to the treatmentgroups being evaluated, normal spines were evaluated using the samemethodology. When all data acquisition was complete, the key was broken,and radiographic, biomechanical, and histologic data were analyzed bytreatment group.

Animal Model:

The sheep lumbar spine model was used because of the biomechanicalsimilarities between the sheep and human lumbar spine. Wilke et al.characterized the biomechanical parameters (range of motion, neutralzone, and level stiffness) of sheep spines and made comparisons withdata from human specimens previously published by White and Panjabi(see, Wilke et al., Spine: 22(20): 2365-2374, 1997; and White AA andPanjabi MM, editors, Clinical Biomechanics of the Spine, 2nd ed., J. B.Lippincott, Philadelphia, Pa., 1990). Wilke et al. found that the“ranges of motion of sheep spines for the different load directions arequalitatively similar in their craniocaudal trends to those of humanspecimens reported in the literature.” They further concluded that:“Based on the biomechanical similarities of the sheep and human spinesdemonstrated in this study, it appears that the sheep spine . . . canserve as an alternative for the evaluation of spinal implants.

Surgical Technique:

Upon arrival at the facility, the 12 sheep were placed in theappropriate pastures of the large animal research barn. They weredewormed and eartagged for identification. Physical examination wasperformed and any animals with signs of respiratory disease had venousblood submitted for a complete blood count (CBC).

The sheep were anesthetized. Wool was removed from the dorsal lumbararea and the sheep positioned in sternal recumbency on the operatingtable.

Iliac Crest Autograft Harvesting: Autograft was used as a control. Thefollowing protocol was followed. The dorsal and dorsolateral lumbar andiliac crest areas were prepared for aseptic surgery with multiple scrubsof povidone-iodine alternated with isopropyl alcohol. The area wasdraped and a 3-cm incision made over the left iliac crest. Followingpartial reflection of the gluteal muscles. An osteotome was used tocreate a small window in the craniodorsal face of the iliac crest. Usinga curette, about 2 cc of autogenous cancellous bone was removed, and waslater packed into one of the implants (e.g. PEEK spacer) used for thelumbar fusion (this is the control case). Intralesional morphine sulfatewas administered prior to closure of the iliac crest incision. The iliaccrest site was closed routinely using 2/0 polysorb for the subcutaneoustissues and stainless steel staples for the skin.

Ventral (“Anterior”) Interbody Fusion: The dorsal and dorsolaterallumbar area was prepared for aseptic surgery with multiple scrubs ofpovidone-iodine alternated with isopropyl alcohol. The area was drapedand a ventrolateral retroperitoneal approach to L3/L4 and L5/L6 throughthe oblique abdominal muscles to the plane ventral to the transverseprocesses was made.

Implant insertion: The bone graft from the iliac crest or a bone graftsubstitute that was being investigated (rhBMP-2+Putty), was placed intothe PEEK spacer (˜1.5 cc of material) and implanted into the disc space,following preparation of the endplates.

Wound Closure: Routine closure of external abdominal muscular fascia (0Polysorb (absorbable suture), subcutaneous tissue (2/0 Polysorb and skin(2/0 monofilament non-absorbable suture) was performed. Operative timefor each animal was usually about 40 minutes. Perioperative antibiotics(Cephazolin sodium) were administered. Postoperative radiographs wereperformed while the sheep were still under general anesthesia.

Aftercare: Immediately after surgery, the sheep were transferred fromthe operating table to a modified wheelbarrow and while still undergeneral anesthesia, taken to a radiology suite where dorsoventral andlateral radiographs of the fusion sites were obtained. Followingradiographic evaluation, while still in the modified wheelbarrow, theywere observed until the swallowing reflex returned. At that point theywere extubated and taken to a trailer where they were propped in sternalrecumbency. At the end of the day, all animals that were operated uponthat day were moved to research pastures. The sheep were housed outdoors(with access to a three-sided shelter) for the convalescence and allowedto exercise at will. Postoperative analgesia was provided as described.The sheep were anesthetized and radiographed at three monthsposoperatively.

Euthanasia: After 6 months postoperatively, the 12 sheep were euthanizedin a humane manner. Euthanasia was performed according to the guidelinesset forth by the AVMA Panel on Euthanasia (J. Am. Vet. Med. Assoc.,202:229-249, 1993). Radiographs of the lumbar fusion sire were taken inthese sheep to evaluate the degree of fusion at L3-L4.

Specimen Collection and Handling: Following euthanasia, a complete grossnecropsy was conducted on all 12 animals. Conventional gross examinationof all major organ systems and histopathological evaluation of anypathological lesions was performed. Animals that died or wereprematurely euthanized during the course of the study had a completenecropsy performed to determine the cause of disease or death. Atnecropsy the lumbar vertebrae that were fused were harvested.

Material Analysis: All samples from the lumbar area from the sheep weresubjected to mechanical testing of the fusion sites. They were testedfor stiffness to saggital and coronal plan bending moments (flexion,extension, right and left lateral bending). As these mechanical testswere nondestructive, the fusion sites were also examined histologically.

Implant Materials:

Twelve treated spinal levels (L4-L5) were evaluated. The study groupsare defined below.

Study Group No. of Samples (N) Autograft Interbody w/PEEK spacer 6(Autograft + PEEK) Inventive Putty Interbody + 6 rhBMP-2 w/PEEK spacer(IP + BMP2 + PEEK) Normal Intact 17 Total 29After the survival phase of the study was completed, the spines wereimmediately frozen for evaluation.

Methods of Analysis: 1. Ex-Vivo Biomechanical Testing of the TreatedLumbar Motion Segment: Flexibility Testing

Unconstrained biomechanical testing was performed in a non-destructivemanner on all spines after the frozen specimens were thawed. All testswere performed within 12 hours of specimen thawing. Specimens were onlyfrozen once. Instrumentation applied to the anterior part of thevertebral body was removed prior to biomechanical testing so that onlythe stiffness of the spine and fusion mass construct was tested, not theinstrumentation. Flexibility of the motion segments was determined inflexion, extension, right and left lateral bending, and right and leftaxial rotation. The purpose of the biomechanical testing was to quantifythe stiffness of the lumbar motion segments augmented with thepreviously described interbody fusion treatments. The treated (L4-L5)motion segments were dissected from the harvested lumbar spines andcleaned of extraneous soft tissues leaving the ligamentous and osseoustissues intact. Specially designed loading and base frames were securedon the L4 and L5 vertebra, respectively.

Moments of 0, 0.5, 2.5, 4.5, 6.5, and 8.5 Nm were achieved in eachloading direction. Static loads were used to apply the pure moments.Three markers reflecting the infrared light were attached to eachvertebra. The locations of the infrared reflective markers will berecorded using three VICON cameras (ViconPeak, Oxford, England) at eachload. Three-dimensional load-displacement data were then acquired withpure moments applied in flexion, extension, left and right lateralbending, and left and right axial rotation. Basic principles of using3-D motion analysis system for investigating the 3-D load-displacementbehavior are well known in the literature.

Biomechanics data from a normal (untreated) intact group of sheep lumbarspine motion segments that have been obtained previously were used asbaseline data for normal lumbar spine motion for L4-L5 in sheep.Differences in the stiffness (flexibility) between groups and thenormals were statistically compared. Non-parametric Kruskal-Wallis andMann-Whitney tests were used to analyze the biomechanics data.

2. Radiographic Assessment:

Radiographs were taken immediately after surgery (AP and lateral views),at regular post-operative intervals (AP and lateral views), and at thetime of sacrifice (AP and lateral views). A high-resolution radiographyunit (Faxitron, Hewlett Packard, McMinnville, Oreg.) and high-resolutionfilm (EKTASCAN B/RA Film 4153, Kodak, Rochester, N.Y.) were used toproduce a high-resolution PA and lateral radiograph of the harvestedlumbar spines after biomechanical testing. Radiographs were scannedusing image analysis software (Image Pro Plus Software v 5.0, MediaCybernetics, Silver Spring, Md.) running on a Windows XP workstation. Avideo camera (Model DFC 280, Leica Microsystems, Cambridge, UK) was usedto acquire the digital images of the radiographs. These radiographs werealso used to gross the samples for histologic analyses as outlinedbelow.

Three blinded evaluators evaluated the resulting Faxitron radiographsfor interbody fusion. On the lateral radiographs, the center of the discspace as well as the anterior and posterior margins were evaluated forfusion based on the following scoring method: 4=continuous bonybridging, 3=increased bone density, 2=lucency with some bony bridging,and 1=non-fusion. Lastly, based on both the P/A and lateral radiographs,the blinded evaluators rated an overall fusion score for the spinallevel using the following criteria:

-   -   3=Solid interbody fusion with no radiolucencies in interbody        space    -   2=Probable fusion with radiolucencies in the interbody space    -   1=Non-fusion with significant radiolucencies in the disc space        with no evidence of superior to inferior bony bridging

3. Undecalcified Histology and Microradiography:

Processing and Stained Undecalcified Sections

In all of the treatment groups, the bisected spinal level was analyzedusing undecalcified techniques (microradiography and multiple stain).Differential staining along with qualitative optical microscopy wasperformed to assess bony bridging and extent of fusion associated withthe autograft or the bone graft substitute packed within the PEEKspacers. Differential staining was used to evaluate the extent of fusionadjacent to and within the peek spacers, the host response to the PEEKspacer and bone graft substitute material (if present), the interface ofthe PEEK spacer, bone graft and substitute incorporation, and boneremodeling within the fusion mass.

After Faxitron radiography, all spinal levels containing an implant weregrossed in the following manner. Using the band saw, a coronal plane cutwas made along the entire length of the spinal column at the anterioraspect of the pedicles leaving anterior tissues intact. Tissuesposterior to the disc space were discarded. Next, the anterior column ofthe spinal level was bisected mid-sagittally to produce right and lefthalves. The entire disc space was left intact. The inferior half of theL4 anterior column adjacent to the treated level was retained. Thesuperior half of the L5 anterior column adjacent to the treated levelwas retained. Right and left sagittal samples from the level were solabeled, fixed in formalin, and processed (sequentially dehydrated inalcohols, cleared in a xylene substitute, and embedded in gradedcatalyzed methyl methacrylate.

After polymerization was complete and the samples hardened, sectioningand staining was performed. The blocks containing the right and lefthalves of the treated aspect of the spinal level were sectioned in thesagittal plane on a low speed diamond saw (Buehler Isomet, Lake Bluff,Ill.). For all embedded tissue blocks, sagittal sectioning commencedfrom the middle of the treated aspect of the spinal level to the lateralaspect of the treated area. Thus, section #1 from the “right block” issampled in the middle of the fusion mass whereas section #6 from the“right block” is sampled at the far lateral aspect of the treated area.Weights were used to produce sections on the order of 300 μm.Approximately 5-10 sections were made in the sagittal plane through eachhalf of the interbody space. If necessary, grinding was performed toobtain the desired thickness. The thickness of the sections was measuredwith a metric micrometer (Fowler, Japan). Differential staining using atrichrome stain was used to permit histological differentiation.

Stained undecalcified sections were scanned using image analysissoftware (Image Pro Plus Software v 5.0, Media Cybernetics, SilverSpring, Md.) running on a Windows XP workstation. A video camera (ModelDFC 280, Leica Microsystems, Cambridge, UK) was used to acquire thedigital images of the stained undecalcified sections.

Section Fusion Criteria: Undecalcified sections were evaluated forfusion in the center of the disc space or thrugrowth region of thedevice, in the anterior margin, and in the posterior margin. Theseanatomic locations for each section were considered to be fused only ifcontinuous bony bridging was found from superior to inferior.

Level Fusion Criteria: Based on all sections evaluated, the followingcriteria were used to determine if histologic fusion was present in thelevel. The spinal level was considered fused if greater than 50% of thesections (corresponding microradiographs were analyzed concurrently butnot “counted twice” for fusion) showed continuous bony bridging. Apartial fusion existed if less than 50% of the sections (andcorresponding microradiographs) showed continuous bony bridging. Anon-fusion existed if none of the sections and correspondingmicroradiographs showed continuous bony bridging.

Microradiography

Undecalcified sections from the treated lumbar spinal levels wereradiographed using a microradiography unit (Faxitron radiography unit,Hewlett Packard, McMinnville, Oreg.) and spectroscopic film (B/RA 4153film, Kodak, Rochester, N.Y.). The thickness of the sections wasmeasured with a metric micrometer (Fowler, Japan) to determine theexposure time. Sections were labeled with ultra-fine permanent markers,placed on the Ektascan B/RA 4153 film, and exposed to the x-ray sourceat 20 kV and 3 mA for approximately 45 seconds for each 100 μm ofsection thickness. The film was then developed, fixed, and analyzed forossification using standard optical microscopy. Microradiographs werescanned using image analysis software (Image Pro Plus Software v 5.0,Media Cybernetics, Silver Spring, Md.) running on a Windows XPworkstation. A video camera (Model DFC 280, Leica Microsystems,Cambridge, UK) was used to acquire the digital images of themicroradiographs.

Analysis of the sections and microradiographs was used to:

-   1) Evaluate the extent of fusion adjacent to and within the peek    spacers, bone graft and substitute incorporation, and bone    remodeling within the fusion mass,-   2) Determine the host response to the biomaterials used, and-   3) Evaluate the interface of the PEEK spacer.

Results 1. Radiographic Evaluation

Good radiographic fusion scores were obtained in both treated groups,with the Inventive Putty+BMP2+PEEK group performing slightly better thanthe autograft group (average 2.8 versus 2.4, Table 1). To study ifrelationships existed between the radiographic fusion score andtreatment, a contingency table was generated (Table 2), and chi-squareanalysis was conducted. As seen in Table 2, the frequency for achievinga fusion score of 3 (Solid Fusion) was 56% for the Autograft+PEEK groupand 83% for the Inventive Putty+BMP2+PEEK group. The frequency forachieving a fusion score of 2 (Probable Fusion) and up was 83% for theAutograft+PEEK group and 100% for the Inventive Putty+BMP2+PEEK group.Chi-square analysis showed that a trend existed (p<0.11) for theInventive Putty+BMP2+PEEK group to achieve higher radiographic fusionscore than the Autograft+PEEK group.

TABLE 1 Average Radiographic Fusion Scores for Each Group TreatmentGroups Fusion Score Autograft + PEEK (n = 6) 2.4 IP + BMP2 + PEEK (n =6) 2.8

TABLE 2 Observed frequencies of overall radiographic fusion scores forthe treatment groups. Total Total Total Percentile Percentile CountCount Count Frequency Frequency for for for for Score for Score Score 3Score 2 Score 1 3 2+ Autograft + PEEK 10 5 3 56%  83% IP + BMP2 + PEEK15 3 0 83% 100%

2. Biomechanics Results

The mean stiffness in the six loading directions for the two treatedinterbody fusion groups as well as an intact normal group is shown inFIGS. 3 through 5. In particular, FIG. 3 provides a chart showing theaverage flexibility of the normal intact and two treated groups inflexion and extension loads. Differences between treated groups and thenormal intact group were statistically significant in both directions.The difference between the two treated groups was significant in flexiononly. FIG. 4 provides a chart showing the average flexibility of thenormal intact and two treated groups in right and left axial rotationloads. Differences between treated groups and the normal intact groupwere statistically significant in both loading directions, whereasdifferences between the two treated groups were not statisticallysignificant. FIG. 5 provides a chart showing the average flexibility ofthe normal intact and two treated groups in right and left lateralbending loads. Differences between treated groups and the normal intactgroup were statistically significant in both loading directions, whereasdifferences between the two treated groups were not statisticallysignificant.

On average, level stiffness of the two treated groups was 3.1(Autograft+PEEK group) and 5.4 (Inventive Putty+BMP2+PEEK group) timesstiffer than the normal intact group in flexion (p<0.0001), 4.6(Autograft+PEEK group) and 6.1 (Inventive Putty+BMP2+PEEK group) timesstiffer than the normal intact group in extension (p<0.0001). Bothtreated groups were 1.7 to 3.0 times stiffer than the normal intactgroup in axial rotation (P<0.0004), and 6.3 to 9.3 stiffer than thenormal intact group in lateral bending (p<0.0001). Comparing flexibilityof the two treated groups, Inventive Putty+BMP2+PEEK group was stifferon average in every loading direction. Statistically significantdifferences were only found in flexion (74% stiffer, p<0.002).

Example 3 Use of Inventive Putty in Posterolateral Fusions

An instrumented ovine posterolateral fusion model was used to evaluatethe ability of Autograft and the Inventive Putty of Example 1+rhBMP-2(IP+rhBMP-2) to effect posterolateral fusion at 6 monthspost-operatively. The efficacy of these treatments to induceposterolateral fusion in the ovine lumbar fusion model was evaluatedusing blinded radiographic, biomechanical, and histologic measures.Assessment of fusion was made with Faxitron high-resolution radiography,non-destructive biomechanical testing, and undecalcified histology withmicroradiography. All analyses were conducted in a blinded fashion. Inaddition, undecalcified histology was used to evaluate theosteocompatibility of the bone graft substitutes. In addition to thetreatment groups being evaluated, biomechanical properties of normalspines were evaluated using the same methodology. When all dataacquisition was complete, the key was broken, and radiographic,biomechanic, and histologic data were analyzed by treatment group.

Animal Model:

The sheep lumbar spine model was used because of the biomechanicalsimilarities between the sheep and human lumbar spine. Wilke et al.characterized the biomechanical parameters (range of motion, neutralzone, and level stiffness) of sheep spines and made comparisons withdata from human specimens previously published by White and Panjabi(see, Wilke et al., Spine: 22(20): 2365-2374, 1997; and White A A andPanjabi M M, editors, Clinical Biomechanics of the Spine, 2nd ed., J. B.Lippincott, Philadelphia, Pa., 1990). Wilke et al. found that the“ranges of motion of sheep spines for the different load directions arequalitatively similar in their craniocaudal trends to those of humanspecimens reported in the literature.” They further concluded that:“Based on the biomechanical similarities of the sheep and human spinesdemonstrated in this study, it appears that the sheep spine . . . canserve as an alternative for the evaluation of spinal implants.

Surgical Technique:

Upon arrival at the facility, the 12 sheep were placed in theappropriate pastures of the large animal research barn. They weredewormed and eartagged for identification. Physical examination wasperformed and any animals with signs of respiratory disease had venousblood submitted for a complete blood count (CBC).

The sheep were anesthetized. Wool was removed from the dorsal lumbararea and the sheep positioned in sternal recumbency on the operatingtable. The dorsal and dorsolateral lumbar area were prepared for asepticsurgery with multiple scrubs of povidone-iodine alternated withisopropyl alcohol. The area was draped and a dorsal approach to L3-L6was made through the dorsal lumbar musculature.

Iliac Crest Autograft Harvesting: Autograft was used as a control. Thefollowing protocol was followed. The dorsal and dorsolateral lumbar andiliac crest areas were prepared for aseptic surgery with multiple scrubsof povidone-iodine alternated with isopropyl alcohol. The area wasdraped and a 3-cm incision made over the left iliac crest. Followingpartial reflection of the gluteal muscles. An osteotome was used tocreate a small window in the craniodorsal face of the iliac crest. Usinga curette, about 2 cc of autogenous cancellous bone was removed, and waslater packed into one of the implants (e.g. PEEK spacer) used for thelumbar fusion (this is the control case). Intralesional morphine sulfatewas administered prior to closure of the iliac crest incision. The iliaccrest site was closed routinely using 2/0 polysorb for the subcutaneoustissues and stainless steel staples for the skin.

Dorsolateral (“posterolateral”) Interbody Fusion: The dorsal lumbar areawas prepared for aseptic surgery with multiple scrubs of povidone-iodinealternated with isopropyl alcohol. The area was draped and localanesthesia (Bupivicaine) was infiltrated along the site of the intendedincision for the dorsal approach to L3 and L4 and spinous processes.

Approach to the transverse processes: A 20 cm. skin incision is made andthe paraspinal muscles are dissected off the spinous processes andlaminae. Facet joints and transverse processes between L3 and L4 areexposed.

Instrumentation and Spine fusion Technique: The transverse processes ofL3 and L4 were decorticated bilaterally. The bone graft from the iliaccrest or a bone graft substitute that is being investigated(rhBMP-2+Inventive Putty), was now placed between the transverseprocesses (˜10 cc per side). The sheep now underwent transpedicularscrew fixation using screws and rods. The pedicle screws and rods wereinserted at this point in the procedure.

Wound Closure: Routine closure of external abdominal muscular fascia (0Polysorb (absorbable suture), subcutaneous tissue (2/0 Polysorb and skin(2/0 monofilament non-absorbable suture) was performed. Operative timefor each animal was usually about 50 minutes. Perioperative antibiotics(Cephazolin sodium) were administered. Postoperative radiographs wereperformed while the sheep were still under general anesthesia.

Aftercare: Immediately after surgery, the sheep were transferred fromthe operating table to a modified wheelbarrow and while still undergeneral anesthesia, taken to the radiology suite where dorsoventral andlateral radiographs of the fusion sites were obtained. Followingradiographic evaluation, while still in the modified wheelbarrow, theywere observed until the swallowing reflex returns. At that point theywere extubated and taken to a trailer where they were propped in sternalrecumbency. At the end of the day, all animals that were operated uponthat day were moved to research pastures. The sheep were housed outdoors(with access to a three-sided shelter) for the convalescence and allowedto exercise at will. Postoperative analgesia was provided as described.The sheep were anesthetized and radiographed at three monthsposoperatively.

Euthanasia: After 6 months postoperatively, the 12 sheep were euthanizedin a humane manner. Euthanasia was performed according to the guidelinesset forth by the AVMA Panel on Euthanasia (J. Am. Vet. Med. Assoc.,202:229-249, 1993). Radiographs of the lumbar fusion sire were taken inthese sheep to evaluate the degree of fusion at L3-L4.

Specimen Collection and Handling: Following euthanasia, a complete grossnecropsy was conducted on all 12 animals. Conventional gross examinationof all major organ systems and histopathological evaluation of anypathological lesions was performed. Animals that died or wereprematurely euthanized during the course of the study had a completenecropsy performed to determine the cause of disease or death. Atnecropsy the lumbar vertebrae that were fused were harvested.

Material Analysis: All samples from the lumbar area from the sheep weresubjected to mechanical testing of the fusion sites. They were testedfor stiffness to saggital and coronal plan bending moments (flexion,extension, right and left lateral bending). As these mechanical testswere nondestructive, the fusion sites were also examined histologically.

Implant Materials:

The study groups are defined below. An animal from the Autograft groupdied prematurely and was excluded from all analyses. Therefore the totalnumber of animals from the Autograft group in the results was reduced to5.

Study Group (per study design) No. of Samples (N) 1) 10 cc/sideAutograft 6 (Autograft) 2) Inventive Putty + rhBMP-2 6 (IP + BMP2) 3)Normal Intact 17 Total 28At the completion of the survival phase of the animal study, the spineswere immediately frozen for evaluation. The efficacy of the bone graftand bone graft substitutes to effect posterolateral fusion and bonyhealing was assessed by performing radiographic, biomechanical, andhistologic analyses as detailed below. The study was performed in ablinded fashion. After all analyses were completed, the key was brokenand radiographic, biomechanical, and histologic data were analyzed bytreatment group.

Methods of Analysis: 1. Radiographic Assessment:

Radiographs were taken immediately after surgery, at regularpost-operative intervals, and at the time of sacrifice. A Faxitron(Hewlett Packard, McMinnville, Oreg.) high-resolution radiography unitand high-resolution film (EKTASCAN B/RA Film 4153, Kodak, Rochester,N.Y.) was used to produce a high-resolution PA radiograph of theharvested lumbar spines after biomechanical testing. Radiographs werescanned using image analysis software (Image Pro Plus Software v 5.0,Media Cybernetics, Silver Spring, Md.) running on a Windows XPworkstation. A video camera (Model DFC 280, Leica Microsystems,Cambridge, UK) was used to acquire the digital images of theradiographs. These radiographs were also used to gross the samples forhistologic analyses as outlined below.

Three blinded evaluators evaluated the resulting Faxitron radiographsfor intertransverse process fusion. On the PA radiograph, on both theright and left sides of the level, the intertransverse process space wasevaluated for fusion based on the following scoring method: 4=continuousbony bridging, 3=increased bone density, 2=lucency with some bonybridging, and 1=non-fusion. Based on both the right and left sides ofthe PA radiographs, the blinded evaluators rated an overall fusion scorefor the spinal level using the following criteria:

-   3=Solid Fusion: Solid intertransverse process fusion on Right AND    Left with no radiolucencies-   2=Possible Fusion: Intertransverse process fusion on the Right OR    Left, but not both. Lucencies in intertransverse process space on    right or left.-   1=Non-Fusion: Isolated bone formation without continuous superior to    inferior bony bridging on both right and left sides. Significant    lucency with no evidence of intertransverse process fusion on the    right or left.    After the treatment code was broken, the radiographic fusion data    were statistically analyzed.

2. Ex-Vivo Biomechanical Testing of the Treated Lumbar Motion Segment:Flexibility Testing:

Unconstrained biomechanical testing was performed in a non-destructivemanner on all spines after the frozen specimens were thawed. Allmetallic posterior instrumentation used to stabilize the posterolateralfusion was removed prior to biomechanical testing so that the stiffnessof the spine and fusion mass construct was tested. Flexibility of themotion segments was determined in flexion, extension, right and leftlateral bending, and right and left axial rotation. All tests wereperformed within 12 hours of specimen thawing. Specimens were onlyfrozen once. The purpose of the biomechanical testing was to quantifythe stiffness of the lumbar motion segments augmented with thepreviously described fusion treatments. The treated (L4-L5) motionsegments were dissected from the harvested lumbar spines and cleaned ofextraneous soft tissue leaving the ligamentous and osseous tissuesintact. Specially designed loading and base frames were secured on theL4 and L5 vertebra, respectively.

Moments of 0, 0.5, 2.5, 4.5, 6.5, and 8.5 Nm were achieved in eachloading direction. Static loads were used to apply the pure moments. Asix-degree of freedom load cell was placed in series with the testedspecimen to verify the applied moments. Three markers reflecting theinfrared light were attached to each vertebra. The locations of theinfrared reflective markers were recorded using three VICON cameras(Vicon Peak, Oxford, England) at each load. Three-dimensionalload-displacement data were then acquired with pure moments applied inflexion, extension, left and right lateral bending, and left and rightaxial rotation. The three-dimensional coordinate data were analyzed toobtain the rotation angles of the superior vertebra with respect to theinferior vertebra and rotational flexibility of each motion segment.

Biomechanics data from a normal (untreated) intact group of sheep lumbarspine motion segments that have been obtained previously were used asbaseline data for normal lumbar spine motion for L4-L5 in sheep.Differences in the stiffness (flexibility) between groups and thenormals were statistically compared. Non-parametric Kruskal-Wallis andMann-Whitney tests were used to analyze the biomechanics data.

3. Undecalcified Histology and Microradiography:

Processing and Stained Undecalcified Sections: In each of the treatmentgroups, the bisected spinal intertransverse process spaces were analyzedusing undecalcified techniques (microradiography and multiple stain).Differential staining along with qualitative optical microscopy wasperformed to assess bony bridging and extent of fusion associated withautograft and the bone graft substitutes. Differential staining was usedto evaluate the host response to the bone graft substitutes.

After Faxitron radiography, all spinal levels containing an implant weregrossed in the following manner. The superior (L3-L4) and inferior(L5-L6) disc spaces were transected leaving the treated (L4-L5)functional spinal unit (FSU) intact. Using the band saw, a coronal planecut was made along the entire length of the spinal column at theanterior aspect of the pedicles leaving posterior tissues intact.Anterior tissues were discarded. Next, the posterior elements of thespinal level were bisected mid-sagittally to produce right and lefthalves. An angled cut in the axial plane was made so that tissuescranial to the cranial transverse processes were discarded on the rightand left sides. An angled cut in the axial plane was made so thattissues caudal to the caudal transverse processes were trimmed anddiscarded on the right and left sides. Tissues in the Right and Leftintertransverse process spaces were further divided in the sagittalplane to produce a medial and lateral sample of the Left fusion mass aswell as a medial and lateral sample of the Right fusion mass. Right andleft medial and lateral samples were so labeled, fixed in formalin, andprocessed (sequentially dehydrated in alcohols, cleared in xylene orxylene substitute, and embedded in graded catalyzed methylmethacrylate).

After polymerization was complete and the samples hardened, sectioningand staining was performed. The blocks containing the transverseprocesses, graft and graft substitutes, and tissues in the transverseprocess space were sectioned in the sagittal plane on a low speeddiamond saw (Buehler Isomet, Lake Bluff, Ill.). For the medial andlateral embedded tissue blocks described above, sectioning commencedfrom the middle of the fusion mass for both the medial and lateralblocks. Thus, section #1 from the “right lateral block” is sampled inthe middle of the fusion mass whereas section #6 from the “right lateralblock” is sampled at the far lateral anatomic aspect of the fusion mass(tips of the transverse processes). Similarly, section #1 from the“right medial block” is sampled in the middle of the fusion mass whereassection #6 from the “right medial block” is sampled at the far medialanatomic aspect of the fusion mass (lamina and facet joints). Weightswere used to produce sections on the order of 300 μm. Approximately 5-10sections were made in the sagittal plane through each half of theintertransverse process space. If necessary, grinding was performed toobtain the desired thickness. The thickness of the sections was measuredwith a metric micrometer (Fowler, Japan). Differential staining using atrichrome stain was used to permit histological differentiation.

Stained undecalcified sections were scanned using image analysissoftware (Image Pro Plus Software v 5.0, Media Cybernetics, SilverSpring, Md.) running on a Windows XP workstation. A video camera (ModelDFC 280, Leica Microsystems, Cambridge, UK) was used to acquire thedigital images of the stained undecalcified sections. Undecalcifiedhistology sections and microradiographs for this study were scanned sothat dorsal was at the top of the image. The ventral side of the sectionwas usually flat and showed two oval transverse processes. Sections werescanned so that transverse processes were at the bottom (ventral aspect)of the image. A mm. scale was scanned at the bottom (ventral aspect) ofthe image. Microsoft Photo editor was used to crop the images.

Section Fusion Criteria: Undecalcified sections were considered fused ifcontinuous bony bridging was found from superior to inferior in thesection. If the presence of non-osseous tissues obviated continuous bonybridging, the section was further evaluated as follows. For non-fusedsections, sections were classified as A) non-fusion with incompletebridge, but with de novo bone found in >50% of the length of thesection, or B) non-fusion with incomplete bridge, with de novo bonefound in <50% of the length of the section.

Right and Left Side Level Fusion Criteria: Based on all sectionsevaluated, the following criteria were used to determine if histologicfusion was present on the right or left side of the level. The right orleft side of the spinal level was considered fused if greater than 50%(>50%) of the sections and corresponding microradiographs showedcontinuous bony bridging. A partial fusion existed if 50% or less (≦50%)of the sections and corresponding microradiographs from the right orleft side of the spinal level showed continuous bony bridging. Anon-fusion existed if none of the sections and correspondingmicroradiographs from the right or left side of the spinal level showedcontinuous bony bridging.

Microradiography: Undecalcified sections from the treated lumbar spinallevels were radiographed using a microradiography unit (Faxitronradiography unit, Hewlett Packard, McMinnville, Oreg.) and spectroscopicfilm (B/RA 4153 film, Kodak, Rochester, N.Y.). The thickness of thesections was measured with a metric micrometer (Fowler, Japan) todetermine the exposure time. Sections were labeled with ultra-finepermanent markers, placed on the Ektascan B/RA 4153 film, and exposed tothe x-ray source at 20 kV and 3 mA for approximately 45 seconds for each100 μm of section thickness. The film was then developed, fixed, andanalyzed for ossification using standard optical microscopy.Microradiographs were scanned using image analysis software (Image ProPlus Software v 5.0, Media Cybernetics, Silver Spring, Md.) running on aWindows XP workstation. A video camera (Model DFC 280, LeicaMicrosystems, Cambridge, UK) was used to acquire the digital images ofthe microradiographs.

Analysis of the sections and microradiographs was used to:

-   1) Evaluate histologic fusion,-   2) Determine the host response to the autograft and bone graft    substitutes, and-   3) Estimate the quality and quantity of bone in the fusion mass    within the intertransverse process space.

Results 1. Radiographic Results

The average radiographic fusion scores for the treated groups arepresented in Table 3. The Inventive Putty+rhBMP-2 group achieved anaverage fusion score of 2.7. The average fusion score for the Autograftgroup was 2.2.

The effect of treatment on the radiographic fusion score was furtheranalyzed. A contingency table was generated (Table 4), and chi-squareanalysis was conducted. As seen in Table 4, the frequency for achievinga fusion score of 3 (Solid Fusion) was 67% for the Inventive Putty+BMP-2group and, 40% for the Autograft group. The frequency for achieving afusion score of 2 (Probable Fusion) and up was 100% for the InventivePutty+BMP-2 group and 80% for the Autograft group. The differencebetween the Inventive Putty+BMP-2 group and the Autograft group was notstatistically significant (p<0.09).

TABLE 3 Average Radiographic Fusion Scores for Each Group TreatmentGroups Fusion Score Autograft (n = 5) 2.2 IP + BMP-2 (n = 6) 2.7

TABLE 4 Observed frequencies of overall radiographic fusion scores forthe treatment groups. Total Total Total Percentile Percentile CountCount Count Frequency Frequency for for for for Score for Score Score 3Score 2 Score 1 3 2+ Autograft 6 6 3 40%  80% IP + BMP-2 12 6 0 67% 100%

2. Biomechanics Results

Group stiffness for the treatment groups for axial rotation, lateralbending, flexion and extension are seen in FIGS. 6 through 8. Inparticular, FIG. 6 provides a chart showing the average stiffness of theintact and treated animal groups under flexion and extension loads, FIG.7 provides a chart showing the average stiffness of the intact andtreated animal groups under axial rotation loads, and FIG. 8 provides achart showing the average stiffness of the intact and treated animalgroups under lateral bending loads. The error bars in these Figuresindicate the standard deviations.

Compared to the normal intact spines, the fusion segments from thetreatment groups were statistically stiffer in all loading directions.Statistical comparison between the two treated groups showed that theonly statistically significant difference was found in the loadingdirection of right lateral bending (Kruskal-Wallis test, p<0.003).Further Mann-Whitney post-hoc tests showed that the flexibility of theInventive Putty+BMP2 treated group in right lateral bending wassignificantly stiffer than the Autograft treated group (87% stiffer,p<0.002).

While the invention has been illustrated and described in detail in thedrawings and foregoing description, the same is to be considered asillustrative and not restrictive in character, it being understood thatonly the preferred embodiment has been shown and described and that allchanges and modifications that come within the spirit of the inventionare desired to be protected. In addition, all references cited hereinare indicative of the level of skill in the art and are herebyincorporated by reference in their entirety.

1. An implantable osteogenic medical material, comprising: a malleable, cohesive, shape-retaining putty comprising 60% to 75% by weight of an aqueous liquid medium and including a bone morphogenic protein incorporated at a level of about 0.6 mg/cc to about 2 mg/cc of the putty; said putty including mineral particles dispersed therein having an average particle diameter in the range of 0.4 mm to 5 mm at a level of 0.25 g/cc to 0.35 g/cc in the putty, the mineral particles comprising a synthetic ceramic; said putty further including insoluble collagen fibers at a level of 0.04 g/cc to 0.1 g/cc of the putty, wherein the mineral particles form a scaffold for bone ingrowth and comprise biphasic calcium phosphate that has a tricalcium phosphate: hydroxyapatite weight ratio of 50:50 to 95:5.
 2. The implantable osteogenic medical material of claim 1, wherein said putty is comprised of about 65% to about 75% of water.
 3. The implantable osteogenic medical material of claim 1, wherein said weight ratio of insoluble collagen fibers to soluble collagen in the putty is in the range of about 75:25 to about 60:40.
 4. The implantable osteogenic medical material of claim 1, wherein said bone morphogenic protein comprises BMP-2.
 5. The implantable osteogenic medical material of claim 4, wherein said BMP-2 comprises recombinant human BMP-2.
 6. The implantable osteogenic medical material of claim 1, wherein said insoluble collagen fibers are present in said putty at a level of 0.05 g/cc to 0.08 g/cc.
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 10. The implantable osteogenic medical material of claim 1, wherein said mineral particles have an average particle diameter in the range of about 0.4 to about 3.0 mm.
 11. The implantable osteogenic medical material of claim 1, wherein said mineral particles have an average particle diameter in the range of about 0.4 to about 2.0 mm.
 12. The implantable osteogenic medical material of claim 1, wherein: said weight ratio of insoluble collagen fibers to soluble collagen is about 75:25 to about 65:35; said mineral particles have an average particle diameter in the range of about 0.4 to about 4.0 mm; said putty includes the insoluble collagen fibers at a level of about 0.05 to 0.08 g/cc; and said bone morphogenic protein is incorporated at a level of about 0.8 mg/cc to about 1.8 mg/cc of the putty.
 13. The implantable osteogenic medical material of claim 12, wherein said bone morphogenic protein comprises recombinant human BMP-2.
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